Bottom Line:
However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive.Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect.These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

ABSTRACTAutophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a mutation, is expressed as a truncated UVRAG(FS) in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAG(FS) abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAG(FS) can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

f7: UVRAGFS inhibits NHEJ repair.(a) Neutral comet assay shows a delay of DNA DSBs repair in UVRAGFS cells. SW480 cells stably expressing empty vector (first row), UVRAGWT (second row) or UVRAGFS (third row) were treated with 1 Gy IR. The DNA damage levels of the cells before IR, 10 min post IR and 24 h post IR were assessed. Representative comet images are shown in the left panel and quantifications are shown on the right. (b) UVRAGFS inhibits UVRAG interaction with Ku70 and Ku80 and the interaction of Ku70/80 with DNA–PKcs. The 293T cells transfected with increasing amounts of Flag-UVRAGFS were treated with IR (5 Gy). WCL were immunoprecipitated with anti-DNA–PKcs or anti-UVRAG, followed by immunoblotting with the indicated antibodies. (c) HEK293 cells stably expressing the EJ5-GFP reporter for NHEJ and the DR-GFP reporter for homologous recombination (HR) were transfected with an empty vector or Flag-UVRAGFS before the induction of DSBs by SceI transfection. The DNA repair activities as assessed by the reconstituted GFP signals were quantified by fluorescence-activated cell sorting. Data shown represent mean±s.d. (n=3). **P<0.01.

Mentions:
We next investigated the possible clinical relevance of UVRAGFS by testing the response of CRC to 5-FU chemotherapy, the first-line treatment for CRC patients, using a tumour xenograft model. Surprisingly, UVRAGFS expression significantly increased tumour sensitivity to 5-FU treatment with an approximate 10-fold reduction in tumour volumes after a 4-week administration of 5-FU (Fig. 6a), compared with a less than twofold reduction in the control group (Fig. 6a–c). Histological analyses revealed a significant reduction in cell proliferation and an increase in the number of cells undergoing apoptosis in 5-FU-treated UVRAGFS tumours, in concordance with induced tumour shrinkage (Fig. 6d). In addition, UVRAGFS expression in CRC cells markedly increased their sensitivity to other DNA-based cytotoxic anticancer agents, including oxaliplatin and irinotecan, as shown by reduced rates of clonogenic survival, whereas UVRAGWT cells were resistant to the drugs (Fig. 6e). To examine the unexpected role of UVRAGFS in tumour chemosensitivity, we measured the levels of γ-H2AX, a sensitive marker of double strand breaks (DSBs)35, and observed that UVRAGFS SW480-tumours accumulated higher levels of γ-H2AX than the controls, which further increased with 5-FU that produces DNA strand breaks (Fig. 6d). Consistent with our observation in xenograft tumours, UVRAGFS expression resulted in a significant increase of γ-H2AX foci and levels in SW480 CRC cells (Supplementary Fig. 7a,b). Furthermore, the overall levels of γ-H2AX were higher in MSI CRC cell lines expressing UVRAGFS compared with the WT counterparts, and likewise, were significantly different between UVRAGFS-positive and -negative primary tumours (Fig. 1b,d). Adding UVRAGWT to UVRAGFS-positive HCT116 and RKO cells at different doses clearly suppressed the levels of DSBs (Supplementary Fig. 7c), highlighting a direct involvement of UVRAGFS in genetic stability. To determine whether the observed accumulation of DSB in UVRAGFS cells reflects impaired DNA repair, we measured unrepaired DSBs after ionizing radiation (IR) using the comet assay. We found that IR induced comparable levels of DNA damage in vector, UVRAGWT and UVRAGFS cells (10 min post-IR in Fig. 7a). However, a high persistence of comet tails was observed 24 h post-irradiation in UVRAGFS cells, whereas UVRAGWT cells have repaired most of the damaged DNA. These data indicate that UVRAGFS disrupts the rapid repair process of DSBs. The inhibitory effect of UVRAGFS on DSB repair was also detected in the autophagy-competent Atg3+/+ and the autophagy- Atg3−/− cells (Supplementary Fig. 7d), suggesting minimal participation of autophagy in the elevated DNA damage induced by UVRAGFS expression.

f7: UVRAGFS inhibits NHEJ repair.(a) Neutral comet assay shows a delay of DNA DSBs repair in UVRAGFS cells. SW480 cells stably expressing empty vector (first row), UVRAGWT (second row) or UVRAGFS (third row) were treated with 1 Gy IR. The DNA damage levels of the cells before IR, 10 min post IR and 24 h post IR were assessed. Representative comet images are shown in the left panel and quantifications are shown on the right. (b) UVRAGFS inhibits UVRAG interaction with Ku70 and Ku80 and the interaction of Ku70/80 with DNA–PKcs. The 293T cells transfected with increasing amounts of Flag-UVRAGFS were treated with IR (5 Gy). WCL were immunoprecipitated with anti-DNA–PKcs or anti-UVRAG, followed by immunoblotting with the indicated antibodies. (c) HEK293 cells stably expressing the EJ5-GFP reporter for NHEJ and the DR-GFP reporter for homologous recombination (HR) were transfected with an empty vector or Flag-UVRAGFS before the induction of DSBs by SceI transfection. The DNA repair activities as assessed by the reconstituted GFP signals were quantified by fluorescence-activated cell sorting. Data shown represent mean±s.d. (n=3). **P<0.01.

Mentions:
We next investigated the possible clinical relevance of UVRAGFS by testing the response of CRC to 5-FU chemotherapy, the first-line treatment for CRC patients, using a tumour xenograft model. Surprisingly, UVRAGFS expression significantly increased tumour sensitivity to 5-FU treatment with an approximate 10-fold reduction in tumour volumes after a 4-week administration of 5-FU (Fig. 6a), compared with a less than twofold reduction in the control group (Fig. 6a–c). Histological analyses revealed a significant reduction in cell proliferation and an increase in the number of cells undergoing apoptosis in 5-FU-treated UVRAGFS tumours, in concordance with induced tumour shrinkage (Fig. 6d). In addition, UVRAGFS expression in CRC cells markedly increased their sensitivity to other DNA-based cytotoxic anticancer agents, including oxaliplatin and irinotecan, as shown by reduced rates of clonogenic survival, whereas UVRAGWT cells were resistant to the drugs (Fig. 6e). To examine the unexpected role of UVRAGFS in tumour chemosensitivity, we measured the levels of γ-H2AX, a sensitive marker of double strand breaks (DSBs)35, and observed that UVRAGFS SW480-tumours accumulated higher levels of γ-H2AX than the controls, which further increased with 5-FU that produces DNA strand breaks (Fig. 6d). Consistent with our observation in xenograft tumours, UVRAGFS expression resulted in a significant increase of γ-H2AX foci and levels in SW480 CRC cells (Supplementary Fig. 7a,b). Furthermore, the overall levels of γ-H2AX were higher in MSI CRC cell lines expressing UVRAGFS compared with the WT counterparts, and likewise, were significantly different between UVRAGFS-positive and -negative primary tumours (Fig. 1b,d). Adding UVRAGWT to UVRAGFS-positive HCT116 and RKO cells at different doses clearly suppressed the levels of DSBs (Supplementary Fig. 7c), highlighting a direct involvement of UVRAGFS in genetic stability. To determine whether the observed accumulation of DSB in UVRAGFS cells reflects impaired DNA repair, we measured unrepaired DSBs after ionizing radiation (IR) using the comet assay. We found that IR induced comparable levels of DNA damage in vector, UVRAGWT and UVRAGFS cells (10 min post-IR in Fig. 7a). However, a high persistence of comet tails was observed 24 h post-irradiation in UVRAGFS cells, whereas UVRAGWT cells have repaired most of the damaged DNA. These data indicate that UVRAGFS disrupts the rapid repair process of DSBs. The inhibitory effect of UVRAGFS on DSB repair was also detected in the autophagy-competent Atg3+/+ and the autophagy- Atg3−/− cells (Supplementary Fig. 7d), suggesting minimal participation of autophagy in the elevated DNA damage induced by UVRAGFS expression.

Bottom Line:
However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive.Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect.These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

ABSTRACTAutophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a mutation, is expressed as a truncated UVRAG(FS) in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAG(FS) abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAG(FS) can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.